The major objective of the proposed research is to investigate the mechanism of the electron-transfer reaction from the covalently-bound acid of the dihydrolipoyl transacetylase component of the multienzyme pyruvate dehydrogenase complex to NAD ion, as mediated by the dihydrolipoyl dehydrogenase (flavoprotein) component. The stoichiometry and kinetics of reduction of the flavoprotein component in the complex by pyruvate plus Coenzyme A and by NADH will be studied by static spectrophotometric and stopped-flow kinetic methods. The results will be compared with the known stoichiometry and kinetics of reduction of isolated dihydrolipoyl dehydrogenase. The kinetics of oxidation of the reduced flavoprotein component of the complex by NAD ion and free lipoic acid derivatives will be studied and compared with isolated lipoyl dehydrogenase. The possible existence of separate binding sites for lipoic acid derivatives and pyridine nucleotides on lipoyl dehydrogenase will be investigated concurrently with the extension of studies which suggest separate binding sites for disulfide substrate and pyridine nucleotides on the mechanistically-similar flavoprotein glutathione reductase. BIBLIOGRAHIC REFERENCES: "Yeast Glutathione Reductase. Steady-State Kinetic Studies of Its Transhydrogenase Activity," G. Moroff, R.S. Ochs, and K.G. Brandt (1976) Arch. Biochem. Biophys., in press (March issue) - preprints enclosed. "Kinetic Studies of the Effect of Adenosine Diphosphoribose on Pig Heart Lipoamide Dehydrogenase," K.G. Brandt and J.K. Lutton, to appear in the published proceedings of the 5th International Symposium on Flavins and Flavoproteins, in press - preprints enclosed.